H2-driven biocatalysis for flavin-dependent ene-reduction in a continuous closed-loop flow system utilizing H2 from water electrolysis

Despite the increasing demand for efficient and sustainable chemical processes, the development of scalable systems using biocatalysis for fine chemical production remains a significant challenge. We have developed a scalable flow system using immobilized enzymes to facilitate flavin-dependent biocatalysis, targeting as a proof-of-concept asymmetric alkene reduction. The system integrates a flavin-dependent Old Yellow Enzyme (OYE) and a soluble hydrogenase to enable H2-driven regeneration of the OYE cofactor FMNH2. Molecular hydrogen was produced by water electrolysis using a proton exchange membrane (PEM) electrolyzer and introduced into the flow system via a designed gas membrane addition module at a high diffusion rate. The flow system shows remarkable stability and reusability, consistently achieving >99% conversion of ketoisophorone to levodione. It also demonstrates versatility and selectivity in reducing various cyclic enones and can be extended to further flavin-based biocatalytic approaches and gas-dependent reactions. This electro-driven continuous flow system, therefore, has significant potential for advancing sustainable processes in fine chemical synthesis.


SH, SH-Tactin activity assay
(1) SH activity assay SH activity assay by H2-dependent reduction of FMN was performed spectrophotometrically as described in (Al-Shameri et al. 2020).
(2) SH-Tactin activity assay 500 mg of Strep-Tactin XT 4Flow resin was loaded with 1.66 mg SH to achieve 3.3 mg g -1 carrier loading.The immobilized SH-Tactin was used to perform H2-dependent reduction of FMN.Activity measurements were performed spectrophotometrically using Agilent Technologies Cary 60 UV-Vis spectrophotometer by monitoring the absorbance decrease of FMN at 500 nm in 2 mL cuvettes after purging with H2.After addition of SH-Tactin, the cuvette was shaken before measurement in the spectrophotometer.The activity of SH-Tactin was measured in 50 mM Tris-HCl pH 8, at 30 °C.The specific activities were calculated using the extinction coefficient of FMN at 500 nm.The extinction coefficient of FMN and FAD at 500 nm ɛ = 2.55 mM -1 cm -1 .

2.2
TsOYE, TsOYE-EziG activity assay (1) TsOYE activity assay Activity measurements of His-tagged TsOYE were performed spectrophotometrically using Agilent Technologies Cary 60 UV-Vis spectrophotometer by monitoring the NADPH absorbance decrease at 365 nm in 2 mL cuvette with 1 mM NADPH and 25 mM cyclohexenone.After addition of TsOYE (6 µg), the cuvette was shaken before measurement in the spectrophotometer.The activity of TsOYE was measured in 50 mM Tris-HCl pH 7.5, at 30 °C.The specific activities were calculated using the extinction coefficient of NADPH at 365 nm.The extinction coefficient of NADH and NADPH at 365 nm ɛ = 3.3 mM -1 cm -1 .
(2) TsOYE-EziG activity assay 50 mg of EziG beads (Amber) was first loaded with 0.72 mg TsOYE to achieve 14.4 mg g -1 carrier loading.This was shaken overnight and used for perform NADPH-dependent reduction of cyclohexenone as described in 2.2-(1).TsOYE-EziG was added by the amount of 0.416 mg for the activity assay.NADPH was chosen instead of FMNH2, due to its ease of control with stoichiometric addition. (

Calculation of enzyme immobilization parameters for corresponding carriers
To determine the applicability of the carriers in immobilizing the biocatalysts, different parameters were assessed.
(1) Immobilization yield Percentage of total enzyme immobilized on the carrier; the yield was assessed by quantifying the protein content in the washing solution via BCA assay under the assumption that any undetected protein was bound to the carrier.

Characterization of immobilized biocatalysts
The strong affinity between the Strep-Tactin matrix and the Strep-tagged protein accounted for the high immobilization yield of 99% (Table S1).The specific activity for flavin reduction of SH in its free form, initially 2.2 U mg -1 , was reduced to 45.5% activity after immobilization.This decrease in activity could be attributed to restricted H2 diffusion and/or fixed flavins/NAD + binding site of the SH, when bound to the Strep-Tactin matrix.Nevertheless, with a protein per carrier loading of 3.3 mg g -1 and an FMN reducing activity per carrier of 3.3 U g -1 (Table S1), the system demonstrated efficacy in flow chemistry applications.Different types of EziG beads varying in hydrophilicity were evaluated for TsOYE immobilization to assess performance (see SI chapter 5).All showed similar immobilization yields at 74-78%.The specific activity of free his-tagged TsOYE was 13.5 U mg -1 .When immobilized in Amber EziG beads (TsOYE-EziG), it showed a relative catalytic activity of 44.8 %.TsOYE-EziG was immobilized with a higher carrier loading (14.4 mg g -1 ) than SH-Tactin, resulting in activity per carrier of 66.9 U g -1 via NADPH (Table S1).
TsOYE-EziG displayed high carrier loading, immobilization yield and relative catalytic activity, comparable to those of OYE3 from Saccharomyces cerevisiae, also immobilized on EziG (100% yield, 52% activity) (Tentori et al. 2020).In contrast, TsOYE immobilized on Celite R-648 showed lower support loading and yield, but remarkable stability in high concentration of organic solvents, suggesting potential for improved stability during biocatalytic reduction of alkenes in micro-aqueous organic solvent (Villa et al. 2023).

TsOYE
EziG Bead, Amber 13.5 ± 0.5 c 44.8 66.9 77.5 ± 1.3 14.4 a Percentage of enzymes bound to the carrier relative to added enzymes.b H2-driven FMN reducing activity.c NADPH-driven cyclohexenone reduction activity by his-tagged TsOYE.A gas addition module encased in steel was designed to facilitate the safe transfer of H2 gas-to-liquid in the flow system (11 cm × Ø 5cm, 150 mL volume).To introduce H2 to the gas addition module, IQS adapters (Ø 6 mm, R 3/8") equipped with mini ball valve were connected to allow pressurized conditions.Pressure gauge was also equipped measure pressure within the gas addition module.Gas addition module was equipped with a 1/4-28 and 10-32 adapter to be connected to the flow system.

Continuous flow setup
To ensure no contamination of atmospheric gas is permeated through the tubes outside the gas addition module during the reaction, Fluran® F-5500-A tubing with very low gas permeability was used.
In addition, the color of the Fluran® tubing was chosen black to block white-light, inhibiting any photoreduction of FMN in aqueous anaerobic conditions (Song et al. 2007;Mifsud et al. 2014).The amount of dissolved H2, dissolved O2 and temperature were measured on-line through an integrated flow sensor to understand the interplay of electrocatalysts and biocatalysts.Modified Clark-type H2 sensor (UNISENSE) was integrated to the flow setup to measure dissolved H2.Optical O2 sensor (PreSens) was integrated with a cuvette attached with an adhesive O2 spot to measure dissolved O2.Rubber septa was inserted over the cuvette, to inhibit any atmospheric air while allowing substrate addition.Also, rubber septa allowed addition of substrate or FMN without addition of oxygen.Redox state of cofactor FMN was measured spectrophotometrically at 500 nm wavelength with a flow cuvette on-line during the reaction.Concentration of oxidised FMN state was measured at 500 nm, due to overlapping of absorbance with cyclohexanone and FMNH2 at 320 nm.The temperature of the flow volume was controlled to 30 °C by portable heating cabinet (HARTMANN).The flow rate was constantly set to 2.6 mL min -1 to emulate gravity flow rate for SH-Strep, unless stated otherwise.The PEM electrolyzer was set to 0.89 A to produce rate of 11 mL min -1 H2 gas and added to the gas addition module.The outlet of the gas addition module was opened and the outflow was checked through bubbling with a syringe with water.• Biotransformation unit is packed with 1.5 mL Strep-Tactin XT 4Flow and loaded with 5 mg of SH, 450 mg of EziG Amber beads were loaded with 6.5 mg of TsOYE as described in 3.1 • Tris-HCl buffer (50 mM, pH 8, 30 °C) is saturated with H2 by the PEM electrolyzer.Flow rate was set to 2.6 mL min -1 using the peristaltic pump.• FMN stock already saturated with H2 is added via the septa to make 1 mM concentration to the flow volume (17 mL) • Observe reduction of FMN to FMNH2 by SH • When H2 is re-saturated, substrate ketoisophorone is added into the cuvette via septa along with DMF as a cosolvent, at a ratio of 2:1.

Electro/-H2 driven biocatalysis (cyclohexenone as substrate)
The experimental procedure is the same as 3.2 except cyclohexenone is added.

3.4
Electro/-H2 driven biocatalysis ((R)-carvone, (S)-carvone as substrate) The experimental procedure is the same as 3.2 except (R)-carvone, (S)-carvone are added as substrate.5 mM concentration is added due to low solubility of carvone to water.The experimental procedure is the same as 3.2 except (R)-carvone and (S)-carvone are added.The gas permeable tubing is changed to PTFE inside the gas addition module due to observation of adsorption of substrate to PVMS tubing.
• The H2 is saturated in the continuous flow volume with the biotransformation unit.The flow rate of the system was increased to 3.3 mL min -1 .• FMN stock is added via the septa to make up 500 µM FMN concentration in the flow volume • Observe reduction of FMN to FMNH2 by SH.
• When H2 is re-saturated, substrate ketoisophorone is added into the cuvette via septa along with DMF as a cosolvent, at a ratio of 2:1.

H2 transfer rate
Figure S3.Transfer of H2 gas to aqueous buffer solutions through a gas-addition module utilizing two different gas-permeable tubing under varying pressure conditions.PVMS tubing (OD = 1.5 mm, ID = 1 mm, length = 2 m), PTFE tubing (OD = 1.5 mm, ID = 1 mm, length = 2 m) H2 gas was transferred to the flow volume (50 mM Tris-HCl buffer, pH 8) by introducing it into the enclosed metal casing of the gas addition module, from where it permeated through the 2-meter gas permeable tubing (PVMS or PTFE).To calculate the H2 transfer from the gas addition module, the dissolved concentration of H2 was observed.This allowed for the quantification of how much H2 was transferred from the gas to liquid phase.The experiment was conducted at room temperature (20 °C).The H2 gas was added to the gas addition module from a pressurized gas cylinder (N5 grade, 99.999% purity).The gas addition module needed to be filled with H2 to reach maximum gas-to-liquid transfer rate.Therefore, when the dissolved H2 concentration reached a plateau, which took less than 5 min, this information was used to calculate H2 transfer rate.For pressurized conditions, the gas output of the gas addition module was closed and the internal pressure was monitored by an integrated pressure gauge.
Under atmospheric pressure, the gas permeable tubes PTFE and PVMS showed gas-to-liquid H2 transfer reaching a concentration of 440 µM and 680 µM dissolved H2, respectively after a single pass through the gas addition module (Figure S3).Under elevated pressure at 1.5 bar, dissolved H2 concentrations reached 640 µM and 1080 µM for PTFE and PVMS, respectively.At 2 bars, the dissolved H2 concentrations of 840 µM and 1180 µM were achieved.With the flow rate (2.6 mL min -1 ) and the volume within the gas permeable tubing (1.57mL), we were able to calculate the contact time of the buffer during the H2 transfer (1.65 min).By dividing the contact time from the dissolved H2 concentration, gas-to-liquid H2 transfer rate was calculated.
Table S2.H2 transfer rate via gas-permeable tubing inside the gas-addition module.The difference in H2 permeability can be ascribed to the variations in their polymeric structures.PVMS has more molecular-level micropores that are created by steric hindrance exerted by the side chains (Özçam et al. 2014).This abundance of micropores leads to higher H2 diffusion rate in PVMS compared to the more rigid structure of PTFE.Also, during the usage of PVMS or gas permeable tubings, pervaporation of substrate or product with high vapor pressure can be expected (Xiao et al. 2006).

Gas
Different membranes have varying properties, including whether it is a multilayer structure, that can influence the permeability of gases and volatile chemicals (Baker and Low 2014), expanding the range of tested membrane will give a comprehensive overview for identifying optimal tubing materials.and incubated for 30 min with shaking.The immobilization yield was calculated by measuring the concentration of supernatant, assuming the rest of TsOYE were immobilized by the carrier.Amber showed highest immobilization yield by 78% followed by Coral 76% and lastly Opal displaying 74%.The functionality for the EMR with entrapped SH and TsOYE in the flow reactor was tested.For electro/-H2 driven biocatalysis in flow setup with EMR, 5 mg of SH and 6.5 mg of TsOYE was added inside the EMR with 30 kDa cellulose membrane (Ultracel®, Merck) on top for entrapment.The rest of the experiment procedure is same as 3.2.The flow volume of the EMR reaction was 25 mL.The conversion of ketoisophorone into levodione was tested in the flow system.The electro-driven biotransformation was performed as described in 3.2.Conversion and enantiomeric excess were measured through GC-FID (see 14.2.2).

Reusability of the immobilized enzymes
Figure S7.The reusability of the immobilized enzymes in production of levodione from ketoisophorone.Repetitions from 2-7 th reactions were conducted in 17 mL conditions (blue).After 7 th reaction, the reaction was started in 300 mL volume (red).
The reusability of the immobilized enzymes (1.5 mL Strep-Tactin XT 4Flow loaded with 5 mg of SH, 450 mg of EziG Amber beads loaded with 6.5 mg of TsOYE) in the flow reactor was tested.The conversion of ketoisophorone to levodione was analysed by GC-FID after multiple runs.Here, the reaction was performed as described in 3.2.Each reaction was performed overnight and the samples were taken 19 h after addition of substrate ketoisophorone.After the reaction was finished, the biotransformation unit was removed from the setup, and the flow system was washed out and equilibrated with a new buffer and a new reaction was started.In the 8 th repetition, 300 mL of volume in a Schott bottle was attached to the flow setup to test bigger scaled reaction.The reaction was stopped at 4% due to presence of O2 in the headspace of the Schott bottle, with the enzymes being inactivated by H2O2 generated by FMNH2.9. Total turnover number for reused immobilized biocatalysts Table S3.Total turnover numbers (TTN   /  )of each biocatalyst based on the sum of products formed after the using the same set of immobilized enzymes.

SH-TTN TsOYE-TTN Reference
After 7 th reuse in flow system ( 17 The conversion of ketoisophorone to levodione was tested in the upscaled flow system.Here, the reaction was performed as described in 3.5.The reaction reached full conversion after 77 h (Figure S8).Faradaic efficiency of the continuous flow system coupled with a commercial PEM electrolyzer was determined with an electro-driven reduction reaction from 3 to 4. The preparation of the experiment is the same as chapter 3.1 except the flow volume (17 mL) of the system was purged of O2 with N2 gas through the gas addition module.Then, FMN and substrate 3 were added to achieve concentrations of 1 mM and 25 mM, respectively.Before turning on the PEM electrolyzer, the N2 in the gas addition module was quickly flushed out with H2 gas from a gas cylinder.This procedure was aimed to minimize the time of electrolysis during which the gas N2 gas within the gas addition module volume (150 mL) transitions to H2. PEM electrolyzer (H-Tec education) was set to 3.3 V, 0.5 A (H2 0.7 mL min -1 ), which was the lowest potential and current to start stable electrolysis of water.The reaction was stopped after 24 hours where 85 % conversion was observed.The Faradaic efficiency was calculated according to the Equation 1. H2 gas that was used to flush the gas addition module volume (150 mL, 1 atm, 20 °C) was also accounted as total electrons passed.The Faradaic efficiency of the continuous flow system for 3 to 4 reaction was calculated to be 0.15 %.

Comparison with literature
13. Environmental impact E factors were roughly calculated to estimate the environmental impact of the reaction (Equation .2) (Sheldon 2017).Ketoisophorone to levodione in 17 mL and upscaled (185 mL) reaction were compared.a Waste component were determined using Tris-HCl buffer at pH 8 (with indicated concentration).No side reactions were observed during this reaction.In entry 2, ketoisophorone mass was calculated based on 69% conversion (based on GC-FID result, 13.1.2).Mass from cultivation, purification step and H2 gas were not included as waste components.
b The amount of waste from buffer and FMN was measured as the reaction were repeated for the immobilized enzymes.c The production of levodione as a product was quantified as the reaction was repeated.For entry 2, 69% product formation was accounted.Theoretical yield was counted for Massproduct calculations.

Figure S1 .
Figure S1.Continuous flow setup with integrated sensors, gas addition module, biotransformation unit, spectrophotometer and PEM electrolyzer.

Figure S2 .
Figure S2.Biotransformation unit packed with biocatalysts and corresponding carriers • First Pack 1.5 mL of Strep-Tactin XT 4Flow resin into the C 10/10 column.Let it settle overnight in 4 °C.• Load SH to the Strep-Tactin XT 4Flow (5 mg for 17 mL scale, 6 mg for 185 mL scale) • Immobilize TsOYE with EziG beads (6.5 mg for 17 mL scale, 8 mg for 185 mL scale).Shake mildly overnight in room temperature.• Load TsOYE-EziG over the SH-Strep inside the column • Add catalase inside the column (amount depending on the reaction) • The biotransformation unit with immobilized biocatalysts are connected to the continuous flow setup 1 bar, flow rate: 2.6 mL min -1

Figure S4 .
Figure S4.Immobilization yield of TsOYE in different types of EziG beads.Average in triplicates, SD is shown.Immobilization yield of TsOYE in different EziG beads via coordinate bonds were investigated.EziG Opal has a hydrophilic surface with pure silica surface and no polymer coating.EziG Coral has a hydrophobic surface with poly(vinylbenzylchloride) coating.EziG Amber has a semi-hydrophilic surface with co-polymer (polystyrene derivative).Purified TsOYE was loaded with respective EziG beads with a carrier loading of 14.4 mg g  −1

Figure S5 .
Figure S5.Continuous flow system for electro-driven FMNH2 biocatalysis with integrated enzyme membrane reactor (EMR) for SH, and TsOYE entrapment.

Figure
Figure S6.(Top) Conversion (%) and enantiomeric excess (ee% R) of substrate ketoisophorone to product levodione by time.(Bottom) Conversions of reactions with reused immobilized biocatalysts.

Figure
Figure S12-B.days after addition of substrate (EMR, 25 mL reaction).Internal standard dodecane peak at 8.069 min.Side product observed at 6.489 min.

Figure
Figure S12-C.days after addition of substrate (EMR, 25 mL reaction).Internal standard dodecane peak at 8.069 min.Side product observed at 6.489 min.

Figure 2 Figure 3 Figure
Figure S17-B.MS spectra of the peak

Table S1 .
Immobilization of enzymes.Activity measurements were performed in triplicates.Mean and standard deviation are shown

Table S4 .
List of parameters and conversions with TsOYE from previous studies in comparison with the current work.Cofactor was compared with other literatures.  /  ) of mol product per mol cofactor

Table S5 .
E factor comparison between ketoisophorone reactions in flow system with different volumes